Biomedical subjects
Publications and source records attributed to L Wu.
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The vascular endothelial cell is generally small and round and has a single small nucleus. It is called a typical endothelial cell (TEC). In human aortas, however, unusually large endothelial cells are often seen. They have multinuclei in a large cytoplasm and are called multinucleated variant endothelial cells (MVECs). MVECs exist individually or in a group, being surrounded by the majority of typical endothelial cells. The number of MVECs is increased with atherosclerosis grade and age. FISH analysis revealed that endothelial cells derived from young subjects had diploid chromosomes. However, aneuploidy was increased with aging in TECs as well as in MVECs, ranging from one to five or even more than five. Expression of LDL receptor on endothelial cells was generally low but greatly elevated in MVECs. Accordingly the uptake of LDL cholesterol was increased when observed in LDL-gold uptake by electron microscopy, whereas those of TECs remained low. These results indicate that human aortic endothelial cells change their characteristics, including shape and even gene, with aging, and that MVECs appear on the human aorta. MVECs actively participate in the development and advancement of atherosclerosis by transporting LDL to the subendothelial intima.
In Dictyostelium discoideum, a unique Gbeta subunit is required for a G protein-coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Galpha2, and Gbeta genes completely impair all these responses. To dissect specificity in Gbetagamma signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gbeta genes and isolated Gbeta alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gbeta subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gbetagamma by making point mutations on Gbeta. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gbetas and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtbetagamma interacts with its regulators, Galpha and phosducin.
Ooplasmic transplantation aimed at restoring normal growth in developmentally compromised oocytes and embryos was evaluated in seven couples (eight cycles) with multiple implantation failures. Two approaches were investigated to transfer ooplasm from donor eggs at metaphase II (MII) stage into patient MII eggs: (i) electrofusion of a ooplasmic donor fragment into each patient egg (three cycles), and (ii) direct injection of a small amount of ooplasm from a donor egg into each patient egg (five cycles). Some donor eggs were used multiple times. Donor eggs were divided into two groups, one being used for ooplasmic extraction and the other one for egg donation. Cleaved embryos resulting from the latter were cryopreserved, where numbers and satisfactory development permitted. A second control group consisted of embryos derived from patient eggs after intracytoplasmic sperm injection without ooplasmic transfer. This was performed when sufficient number of eggs were available (n = 5). Donor eggs (n = 40) were evaluated cytogenetically after micromanipulation in order to confirm the presence of chromosomes. One egg was anuclear and the recipient embryos were not transferred. Normal fertilization was significantly higher after injection of ooplasm (63%) in comparison with fusion (23%). Pronuclear anomalies appeared enhanced after fusion with ooplasts. Embryo morphology was not improved in the three cycles with electrofusion and patients did not become pregnant. An improvement in embryo morphology was noted in two patients after injection of ooplasm and both became pregnant, but one miscarried. A third pregnancy was established in the repeat patient, without obvious embryo improvement. One baby was born and the third pregnancy is ongoing with a normal karyotype. Two other patients with male factor infertility had poor embryos after ooplasmic injection, but the donor embryo controls were also poor. The patients did not become pregnant and had no donor embryos frozen. Ooplasmic transfer at the MII stage may be promising in patients with compromised embryos; however, evaluation of ooplasmic anomalies and optimization of techniques will require further investigation prior to widescale application.
The Ped (preimplantation embryonic development) gene influences the rate of mouse preimplantation embryonic development and subsequent survival. Four similar tandem genes in the Q region of the major histocompatibility complex-Q6, Q7, Q8, and Q9-were identified as Ped gene candidates. In this study, expression of these genes during preimplantation development was examined and quantitated by reverse transcription-polymerase chain reaction and single nucleotide primer extension assays in order to investigate their contribution to the Ped gene phenotype. The Q7/Q9 gene pair was found to be transcribed in preimplantation mouse embryos, whereas transcription of the Q6/Q8 gene pair was undetectable. Both Q7 and Q9 are expressed in embryos from one Ped fast strain, C57BL/6, while only the Q9 gene is expressed in another Ped fast strain, B6.K2. These results suggest that both the Q7 and Q9 genes can function as the Ped gene in the mouse. Interestingly, the expression pattern of the Q7 and Q9 genes in preimplantation embryos is the same as in splenic lymphocytes. However, the Q6 and Q8 genes are expressed in splenic lymphocytes but not in preimplantation embryos. Treatment of mouse preimplantation embryos with interferon gamma (gamma-IFN) did not induce expression of the Q6/Q8 genes but enhanced expression of the Q7/Q9 genes. The mechanism of this differential transcription pattern is currently under investigation.
OBJECTIVE: To test the hypothesis that the enhanced inhibition by melatonin of the norepinephrine-induced vasoconstriction and formation of inositol phosphate in spontaneously hypertensive rats are mediated by its antioxidant effect METHODS: Aortic rings from spontaneously hypertensive rats and age-matched Wistar-Kyoto rats were used for measuring vascular contraction forces. Cultured aortic smooth muscle cells were prelabelled with myo-[2-3H]-inositol for evaluation of formation of inositol phosphate after exposure to agonist or antagonist Basal or forskolin-induced formation of cyclic AMP was evaluated using a [3H]-cyclic AMP assay system. Oxygen-derived free radicals were generated with a hypoxanthine and xanthine oxidase system. RESULTS: The inhibition of the norepinephrine-induced aortic contraction by melatonin was more potent in spontaneously hypertensive than it was in Wistar-Kyoto rats. The inhibition of the norepinephrine-induced formation of inositol phosphate by 0.3-300 micromol/l melatonin was also greater in smooth muscle cells from spontaneously hypertensive rats than it was in those cells from Wistar-Kyoto rats. In contrast, the inhibition of the norepinephrine-induced formation of inositol phosphate in smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto rats by 2-iodomelatonin, an agonist of melatonin receptors, was not different. Prazosin, but not yohimbine, eliminated or partially inhibited the norepinephrine-induced formation of inositol phosphate in smooth muscle cells from Wistar-Kyoto rats or from spontaneously hypertensive rats, respectively. In the presence both of prazosin and of melatonin, the norepinephrine-induced production of inositol phosphate was abolished in smooth muscle cells from spontaneously hypertensive rats. Furthermore, superoxide dismutase significantly inhibited the norepinephrine-induced aortic contraction and formation of inositol phosphate in smooth muscle cells from spontaneously hypertensive rats, but not in those cells from Wistar-Kyoto rats. In contrast, catalase had no effect on the norepinephrine-induced formation of inositol phosphate and vascular contraction either in cells from spontaneously hypertensive rats or in cells from Wistar-Kyoto rats. Hypoxanthine-xanthine oxidase induced formation of more inositol phosphate in smooth muscle cells from spontaneously hypertensive rats than it did in those from Wistar-Kyoto rats. Melatonin and superoxide dismutase similarly inhibited the hypoxanthine-xanthine oxidase-induced formation of inositol phosphate more in cells from spontaneously hypertensive rats than it did in those from Wistar-Kyoto rats. However, melatonin had no effect either on basal or on the forskolin-induced formation of cyclic AMP in smooth muscle cells from rats of both strains. CONCLUSION: The enhanced inhibitory effect of melatonin on the norepinephrine-induced cellular production of inositol phosphate in spontaneously hypertensive rats was not mediated by melatonin receptors or alpha-adrenoceptors. Rather, the antioxidant effect of melatonin could become important in spontaneously hypertensive rats, which are suspected to have a lower cellular content of antioxidants or a greater sensitivity to superoxide anions, or both, of the vascular tissue.
OBJECTIVE: The purpose of this study was to assess the role of transforming growth factor (TGF)-beta1 in the development of diabetes-associated mesenteric vascular hypertrophy and in the antitrophic effect of angiotensin converting enzyme inhibitors. DESIGN AND METHODS: Streptozotocin-induced diabetic and control Sprague-Dawley rats were randomly allocated to treatment with the angiotensin converting enzyme inhibitor ramipril or to no treatment and were killed 1 or 3 weeks after the streptozotocin injection. Blood was collected and mesenteric vessels removed. Mesenteric vascular weight was measured and TGF-beta1 and alpha1 (type IV) collagen messenger (m)RNA levels were analysed by Northern analysis. Immunohistochemical analyses for TGF-beta1 and type IV collagen were also performed. RESULTS: The diabetic rats had increased mesenteric vessel weight at 3 weeks but not at 1 week and a concomitant rise in mesenteric TGF-beta1 and in alpha1 (type IV) collagen mRNA levels. Ramipril treatment attenuated mesenteric vessel hypertrophy and prevented the increase in TGF-beta1 and alpha1 (type IV) collagen mRNA levels after 3 weeks of diabetes. The immunohistochemical analysis revealed that diabetes was associated with increased TGF-beta1 and type IV collagen protein and extracellular matrix accumulation in mesenteric vessels, and this increase was reduced by ramipril treatment. CONCLUSIONS: These results support the concept that TGF-beta is involved in the changes associated with diabetic vascular disease, and suggest a mechanism by which angiotensin converting enzyme inhibitors exert their antitrophic effects.
Thymic dendritic cells (DC) mediate negative selection at a relatively late stage of the T-cell developmental pathway. We present evidence that the development of thymic DC and of T-lineage cells is linked via a common precursor at an early stage of thymocyte development. T-lineage precursor populations from the adult mouse thymus, prior to T-cell receptor gene rearrangement, display a capacity to produce DC as well as T cells in the thymus, and are very efficient precursors of DC in culture. These lymphoid/DC precursors have little capacity to form myeloid cells, indicating that thymic DC are a lymphoid-related rather than myeloid-related lineage. In contrast to myeloid-related DC, granulocyte-macrophage colony-stimulating factor is not required for the development of these lymphoid-related DC in vivo or in vitro. DC can develop in mutant mice lacking mature T cells, provided the common precursors are present. However, in mutant mice lacking functional Ikaros transcription factors, there are deficiencies in lymphoid precursor cells, in mature lymphoid cells and in DC.
A new commercial glycoprotein G-based enzyme immunoassay (gG-EIA) was compared with Western blotting (WB) for detection of herpes simplex virus type 1 (HSV-1) or HSV-2 type-specific antibodies in 193 serum samples. Sensitivity for HSV-1 was 95%; specificity was 96%. Sensitivity for HSV-2 was 98%; specificity was 97%. Twelve of 13 serum samples with equivocal gG-EIA results were serotyped by WB.
The CC-chemokine receptor CCR5 is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of CD4+ cells and interacts directly with the viral surface glycoprotein gp120. Although receptor chimera studies have provided useful information, the domains of CCR5 that function for HIV-1 entry, including the site of gp120 interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of CCR5 to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish CCR5-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair Env-mediated membrane fusion and the gp120-CCR5 interaction. Of these three residues, only D11 is necessary for CC-chemokine-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular CCR5 residues. Thus, the gp120 and CC-chemokine binding sites on CCR5 are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal CCR5 domain.
We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1alpha against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1beta, MIP-1alpha. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1beta, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1beta, MIP-1alpha.
The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1BaL) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.
Human immunodeficiency virus type 1 (HIV-1) requires the presence of specific chemokine receptors in addition to CD4 to enter target cells. The chemokine receptor CCR5 is used by the macrophage-tropic strains of HIV-1 that predominate during the asymptomatic stages of infection. Here we identify a small tyrosine-rich region of CCR5 proximal to the N-terminal cysteine that is critical for entry of macrophage-tropic and dual-tropic variants of HIV-1. HIV-1 infection of cells expressing CCR5 mutants with changes in this region was substantially reduced compared with the infection of cells bearing wild-type CCR5. Simian immunodeficiency virus (SIVmac239) entry was also ablated on a subset of these mutants but enhanced on others. These differences in virus entry were correlated with the relative ability of soluble, monomeric HIV-1 and SIVmac239 gp120 glycoproteins to bind the CCR5 mutants. These results identify a region of CCR5 that is necessary for the physical association of the gp120 envelope glycoprotein with CCR5 and for HIV-1 infection.
A retrospective study of DNA flow cytometry (FCM) in paraffin-embedded tissues of urinary bladder transitional cell carcinoma (TCC) was performed on 239 biopsy samples taken from 81 patients in the period from 1984 to 1994. 210 (87%) were analysable. Of these samples 21 patients had multiple biopsies taken from large tumours and/or bladder mucosa showing an endoscopically normal appearance. DNA-FCM results have been evaluated comparing ploidy and histopathological grade, clinical stage and different clinical status, i.e., first diagnosis, recurrence and patients who died from bladder cancer. Our results indicate that 'diploid' FCM correlated with a better prognosis, whilst DNA aneuploid correlated with malignancy and a poorer prognosis. There was a trend to an increasing incidence of DNA aneuploidy as the grade of the tumour rose and the proportion of biopsies with aneuploidy was significantly higher in malignant tissue samples, recurrences and in biopsies from patients who died from TCC than in other groups. In 12 patients from whom several biopsies were obtained, samples from recurrences had significantly higher DNA aneuploidy than those from the first diagnosis.
GLUT2 is the major glucose transporter in pancreatic beta-cells and hepatocytes. It plays an important role in insulin secretion from beta-cells and glucose metabolism in hepatocytes. To better understand the molecular determinants for GLUT2's distinctive glucose affinity and its ability to transport fructose, we constructed a series of chimeric GLUT2/GLUT3 proteins and analyzed them in both Xenopus oocytes and mammalian cells. The results showed the following. 1) GLUT3/GLUT2 chimera containing a region from transmembrane segment 9 to part of the COOH-terminus of GLUT2 had Km values for 3-O-methylglucose similar to those of wild-type GLUT2. Further narrowing of the GLUT2 component in the chimeric GLUTs lowered the Km values to those of wild-type GLUT3. 2) GLUT3/GLUT2 chimera containing a region from transmembrane segment 7 to part of the COOH-terminus of GLUT2 retained the ability to transport fructose. Further narrowing of this region in the chimeric GLUTs resulted in a complete loss of the fructose transport ability. 3) Chimeric GLUTs with the NH2-terminal portion of GLUT2 were unable to express glucose transporter proteins in either Xenopus oocytes or mammalian RIN 1046-38 cells. These results indicate that amino acid sequences in transmembrane segments 9-12 are primarily responsible for GLUT2's distinctive glucose affinity, whereas amino acid sequences in transmembrane segments 7-8 enable GLUT2 to transport fructose. In addition, certain region(s) of the amino-terminus of GLUT2 impose strict structural requirements on the carboxy-terminus of the glucose transporter protein. Interactions between these regions and the carboxy-terminus of GLUT2 are essential for GLUT2 expression.
We investigated the effect of 2 months of exposure to cold conditions (0-5 C) on capillarization and on fiber size, distribution and ultrastructure in the pectoralis muscle of nine pigeons (Columbia livia; mean body mass 700 31 g) and compared the results with measurements from four control birds (mean mass 715 42 g) kept at normal ambient temperature (22-23 C) for the same period. Superficial and deep portions of the muscles, taken from the central area of the right or left pectoralis major muscle, were perfusion-fixed in situ, processed for electron microscopy and analyzed by morphometry. Aerobic fibers represented the vast majority of fibers (93 1 %, mean s.e.m.) in all samples. After cold-acclimation, fiber sectional area was reduced and capillary density increased proportionally. There was no change in the degree of orientation (anisotropy) of capillaries, capillary-to-fiber ratio or fiber type distribution compared with controls. The volume density of mitochondria and lipid droplets in aerobic fibers and capillary diameter increased in response to cold, while the linear relationship between capillary length per fiber volume and fiber mitochondrial volume density remained unchanged. Capillary surface area, intrafiber lipid deposition and fiber mitochondrial volume density were all correlated in cold-acclimated pigeons. The results indicate a close match between the aerobic capacity of the highly aerobic fibers of the pectoralis muscle and their vascularization to meet the increased energetic demand of shivering.
Multinucleated variant endothelial cells (MVECs) generally exist in atherosclerotic human aorta and even in nonatherosclerotic aorta. Because the number of nuclei is increased in every MVEC, and because DNA instability was suspected, a series of oncogene expressions was conducted to clarify the nature of nuclear abnormality. The tumor suppressor gene p53 was found to be specifically expressed in the multinuclei of MVECs, while double nuclei were sometimes positive, and mononuclear typical endothelial cells were always negative for p53. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) revealed extra bands in exons 5 and 7 of the p53 gene, but no additional band in exons 6 and 8. In a BCL family, BCL-2 was coexpressed in one or two nuclei in the perinuclear space of the multinuclei of MVECs, whereas MCL-1, BCL-XS/L, and BAX were all negative, indicating that the BCL-2 coding gene is expressed only in the corresponding one or two nuclei of the multinuclei. Another oncogene, c-MET (hepatocyte growth factor receptor), was universally expressed in either type of endothelial cells, but other oncogenes, k-RAS and c-ERBB2, were not expressed in either type. MVECs were derived from human aorta and therefore non-tumorous somatic cells. No morphologic evidence of apoptosis was found. Although it is unclear that the extra bands came from the MVECs or just from ECs associated with atherosclerosis, combined immunocytological studies and PCR analysis suggest that MVECs express mutant type p53.
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